As highlighted in the 'Bad Bugs, No Drugs' campaign by the Infectious Diseases Society of America, There simply aren't enough new drugs in the pharmaceutical pipeline to keep pace with drug-resistant bacterial infections, so-called 'superbugs'. Numerous hospitals worldwide have experienced outbreaks caused by multidrug-resistant (MDR) Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae, three of the 6 top-priority dangerous ESKAPE pathogens that require the most urgent attention to discover new antibiotics. Sadly, no novel antibiotics against these Gram-negative 'superbugs' will be available for many years to come. Polymyxins (i.e. polymyxin B and colistin) are now being used as the last-line therapy for these very problematic MDR pathogens. Most unfortunately, the emergence of polymyxin resistance has been increasingly reported recently. In essence, resistance to polymyxins implies a total lack of antibiotics for treatment of life-threatening infections caused by these pandrug-resistant (PDR) Gram-negative bacteria. Research Design: This project will apply systems pharmacology to antibiotic pharmacokinetics/pharmacodynamics (PK/PD) to identify novel polymyxin combinations with FDA-approved nonantibiotic drugs against PDR P. aeruginosa, A. baumannii and K. pneumoniae. Our over-arching hypothesis is that the identified polymyxin-nonantibiotic combinations will synergistically kill Gram-negative 'superbugs' with minimal development of resistance, and integration of systems pharmacology with antimicrobial PK/PD modeling will elucidate the underlying mechanism(s) of the synergistic interaction between host-pathogen-drug. Our specific aims are to: (1) Identify novel polymyxin-nonantibiotic combinations against pandrug-resistant P. aeruginosa, A. baumannii and K. pneumoniae; (2) Investigate the in vitro PK/PD of active polymyxin-nonantibiotic combinations; (3) Evaluate the synergistic killing and suppression of polymyxin resistance by the active combinations over 2-week treatment using hollow fiber infection model and conduct omics studies; (4) Demonstrate proof-of-concept for the most active combinations using mouse infection models and conduct integrated omics studies to understand the host-pathogen-drug interactions; and (5) Conduct systems pharmacology network analysis and PK/PD mathematical modeling on the polymyxin- nonantibiotic combinations. Transcriptomics, proteomics and metabolomics will be utilized to investigate the interaction between host, pathogen and drug. Together, these studies will identify the best polymyxin-nonantibiotic combination (plus one backup) for further pharmacological evaluations. Significance: This project holds great promise for development of novel polymyxin combinations. As all drugs have already been approved by FDA, our multi-disciplinary approach will provide a fast-track and cost-effective solution to combat these very problematic Gram-negative 'superbugs'. Overall, this project targets the urgent global unmet medical need, lack of new antibiotics.